This invention relates to the use of enzymes in cleaning applications, especially in household cleaning applications. For this purpose it is known to use, for example, proteases, lipases, amylases and cellulases.
However, these enzymes are incapable of removing all kinds of dirt, soil or stains present on or in textiles, on kitchenware, etc., as are synthetic detergents and other components of cleaning compositions known in the art.
For instance, stains of e.g. vegetable origin are not sufficiently removed by current detergents, if at all.
Usually detergents comprise a bleaching agent which, through oxidative reactions, decolourizes the stains, but does not remove them.
Moreover, these bleaching agents may cause damage to the object to be cleaned, especially when it has to be cleaned often.
Stains are usually defined as intensively coloured substances that colour a fabric even when they are present in very small amounts on fibres and resist removal by detergents alone (Cutler W G, Kissa E, 1987, Detergency, theory and technology, Chapter 1, p 1-90).
A common type of stain originates from vegetable materials including the associated pigments. Examples of such stains are grass, vegetables such as spinach, beetroot, carrot, tomatoes, fruits such as all types of cherries and berries, peach, apricot, mango, bananas and grapes as well as stains from drinks derived from plant material, such as wine, beer, fruit juices and additionally tomato sauce, jellies, etc.
Pigments in these vegetable materials are usually associated with the fibrous materials which are a major part of the plant cell walls, either via covalent bonds or via physical binding (xe2x80x9cstickingxe2x80x9d). Removal of these pigments can be very difficult, since detergents can barely remove the fibre-pigment mass from a surface to be cleaned. Recent research has shown that plant cell walls consist of a complicated network of fibrous materials. The composition of the cell walls varies considerably, depending on the source of the vegetable material. However, in general its composition can be summarized as mainly comprising non-starch polysaccharides. These polysaccharides can be found in various forms: cellulose, hemicellulose and pectins.
The composition of a plant cell wall is both complex and variable. Polysaccharides are mainly found in the form of long chains of cellulose (the main structural component of the plant cell wall), hemicellulose (comprising e.g. various xcex2-xylan chains) and pectin. The occurrence, distribution and structural features of plant cell wall polysaccharides are determined by: 1. plant species; 2. variety; 3. tissue type; 4. growth conditions; and 5. ageing (Chesson (1987), Recent Advances in Animal Food Nutrition, Haresign on Cole, eds.). Butterworth, London, 71-89).
Basic differences exist between monocotyledons (e.g. cereals and grasses) and dicotyledons (e.g. clover, rapeseed and soybean) and between the seed and vegetative parts of the plant (Carre{acute over ( )} and Brillouet (1986), Science and Food Agric. 37, 341-351). Monocotyledons are characterized by the presence of an arabinoxylan complex as the major hemicellulose backbone. The main structure of hemicellulose in dicotyledons is a xyloglucan complex. Moreover, higher pectin concentrations are found in dicotyledons than in monocotyledons. Seeds are generally very high in pectic substances, but relatively low in cellulosic material. Three more or less interacting polysaccharide structures can be distinguished in the cell wall:
1. the middle lamella forms the exterior cell wall. It also serves as the point of attachment for the individual cells to one another within the plant tissue matrix. The middle lamella consists primarily of calcium salts of highly esterified pectins;
2. the primary wall is situated just inside the middle lamella. It is a well-organized structure of cellulose microfibrils embedded in an amorphous matrix of pectin, hemicellulose, phenolic esters and proteins;
3. the secondary wall is formed as the plant matures.
During the plant""s growth and ageing phase, cellulose microfibrils, hemicellulose and lignin are deposited.
Until the present invention there was no detergent or other cleaning agent available capable of breaking down the complex fibrous structure or gel-like matrix of plant cell walls or components thereof, thereby releasing the pigment from the surface, object, or fabric to be cleaned.
The present invention not only seeks to solve the problem of removing stains of vegetable origin, but it also aims to help remove soil and dirt, which soil and dirt have, at least in part, a similar structure (e.g. stains of a food composition in which plant cell wall components are present as thickeners or gelating agents or the like).
The present invention can thus solve this problem by providing a cleaning composition comprising at least one plant cell wall degrading enzyme, or a substance having the same activity as such an enzyme, with the proviso that when only one type of such an enzyme is present, it is not a cellulase. Thus the invention does not contemplate the use of solely one or more cellulases alone, but employs other plant cell wall degrading enzymes (although cellulases can be included with such other enzymes if desired). Hence a first aspect of the invention relates to a cleaning composition comprising one or more substances that are capable of degrading plant cell walls, other than a composition comprising one or more cellulases as the only plant cell wall degrading substance(s).
This proviso is made because cellulases are known to be included in cleaning compositions. In current detergents intended for cleaning textiles cellulases are sometimes incorporated to improve softness, as an anti-pilling component, or for additional cleaning effects. Cellulases can, however, not be used in significant amounts, since many textile fibres comprise a high percentage of cellulose fibres, which of course are susceptible to breakdown by these enzymes. These enzymes by themselves are therefore not particularly suitable for the main purpose of the present invention, since they cannot be added in a sufficient amount to remove stains of vegetable origin without damaging the textile. They can, however, be used in combination with other enzymes which are capable of breaking down cell walls, in which case they can be added in lower amounts, because of the concerted action on the fibrous mass of such stains by the mixture of enzymes. Thus, the use of cell wall degrading enzymes can create optimal cleaning conditions, without damage to textile fibres, if the amount of cellulase(s) is reduced to less than 50%, preferably less than 25% and most preferably less than 10% of the total amount (w/w) of plant cell wall degrading enzymes added. In some embodiments there may be no cellulase(s) at all.
Cleaning compositions according to the invention thus comprise at least 50%, preferably at least 75% of a pectinase and/or a hemicellulase based on the total amount (w/w) of plant cell wall degrading enzymes. In some embodiments the composition may comprise 90% (w/w) or more of a pectinase or a hemicellulase as the plant cell wall degrading enzyme activity.
There is a high degree of interaction between cellulose, hemicellulose and pectin in the cell wall. The enzymatic degradation of these rather intensively cross-linked polysaccharide structures is not a simple process. A large number of enzymes are known to be involved in the degradation of plant cell walls. They can broadly be subdivided in cellulases, hemicellulases and pectinases (Ward and Young (1989), CRC Critical Rev. in Biotech. 8, 237-274).
Cellulose is the major polysaccharide component of plant cell walls. It consists of xcex21,4 linked glucose polymers.
Cellulose can be broken down by cellulases, also called cellulolytic enzymes. Cellulolytic enzymes have been divided traditionally into three classes:
endoglucanases,
exoglucanases or cellobiohydrolases and xcex2-glucosidases
(Knowles, J., et al. (1987), TIBTECH 5, 255-261). Like all cell wall degrading enzymes they can be produced by a large number of bacteria, yeasts and fungi. Apart from cellulases degrading xcex2-1,4 glucose polymers, endo-1,3/1,4 xcex2-glucanases and xyloglucanases should be mentioned (Ward and Young op. cit.).
Pectins are major constituents of the cell walls of edible parts of fruits and vegetables. The middle lamella which are situated between the cell walls are mainly built up from protopectin which is the insoluble form of pectin. Pectins are considered as intracellular adhesives and due to their colloidal nature they also have an important function in the water regulation system of plants. The amount of pectin can be very high. For example, lemon peels are reported to contain pectin at up to 30% of their dry weight, orange peels contain from 15-20% and apple peels about 10% (Norz, K. (1985). Zucker und Susswaren Wirtschaft 38, 5-6).
Pectins are composed of a rhamno-galacturonan backbone in which 1,4-linked (xcex1-D-galacturonan chains are interrupted at intervals by the insertion of 1,2-linked (xcex1-L-rhamnopyranosyl residues (Pilnik, W. and A. Voragen (1970), In: The Biochemistry of fruits and their products, vol. 1, chapter 3, p. 53. Acad. Press). Other sugars, such as D-galactose, L-arabinose and D-xylose, are present as side chains. A large part of the galacturonan residues is esterified with methyl groups at the C2 and C3 position.
A large number of enzymes are known to degrade pectins. Examples of such enzymes are pectin esterase, pectin lyase (also called pectin transeliminase), pectate lyase, and endo- or exo-polygalacturonase (Pilnik and Voragen (1990). Food Biotech 4, 319-328). Apart from enzymes degrading smooth regions, enzymes degrading hairy regions such as rhamnogalacturonase and accesory enzymes have also been found (Schols et al. (1990), Carbohydrate Res. 206, 105-115; Searle Van Leeuwen et al. (1992). Appl. Microbiol. Biotechn. 38, 347-349).
Hemicelluloses are the most complex group of non-starch polysaccharides in the plant cell wall. They consist of polymers of xylose, arabinose, galactose or mannose which are often highly branched and connected to other cell wall structures. Thus a multitude of enzymes is needed to degrade these structures (Ward and Young op.cit.). Xylanase, galactanase, arabinanase, lichenase and mannanase are some hemicellulose degrading enzymes.
Endo- and exo-xylanases and accessory enzymes such as glucuronidases, arabinofuranosidases, acetyl xylan esterase and ferulic acid or coumaric acid esterase have been summarized by Kormelink (1992, Ph.D.-thesis, University of Wageningen, The Netherlands). They are produced by a wide variety of micro-organisms and have varying temperature and pH optima.
Like other cell wall degrading enzymes (CWDE""S) galactanases occur in many micro-organisms (Dekker and Richards (1976), Adv. Carbohydrat. Chem. Biochem. 32, 278-319). In plant cell walls two types of arabinogalactans are present: type I 1,4 xcex2-galactans and type II 1,3/1,6 xcex2-galactans which have a branched backbone (Stephen (1983). In: The Polysaccharides. G. O. Aspinael (ed.). Ac. Press, New York, pp. 97-193). Both types of galactans require their own type of endo enzyme to be degraded. It can be expected that other enzymes, such as arabinan-degrading enzymes and exo-galactanases play a role in the degradation of arabinogalactans.
The hemicellulose 1,3-1,4-xcex2-glucan is a cell wall component present in cereal (barley, oat, wheat and rye) endosperm. The amount of xcex2-glucan in cereal endosperm varies between 0.7-8%. It is an unbranched polysaccharide built from cellotriose and cellotetraose residues linked by a 1,3-glucosidic bond. The ratio tri/tetra saccharose lies between 1.9 and 3.5.
Lichenase (EC 3.2.1.73) hydrolyse 1,4-beta-D-glucosidic linkages in beta-D-glucans containing 1,3- and 1,4-bonds. Lichenase reacts not on beta-D-glucans containing only 1,4-bonds such as for example in cellulose. Thus, damage of cellulose fibres in fabrics does not occur by the application of lichenase. Lichenases are produced by bacteria like B. amyloliquefaciens, B. circulans, B. licheniformis and plants (Bielecki S. et al. Crit. Rev. in Biotechn. 10(4), 1991, 275-304).
Arabinans consist of a main chain of xcex1-L-arabinose subunits linked (xcex1-(1xe2x86x925) to another. Side chains are linked xcex1-(1xe2x86x923) or sometimes xcex1-(1xe2x86x922) to the main xcex1-(1xe2x86x925)-L-arabinan backbone. In apple, for example, one third of the total arabinose is present in the side chains. The molecular weight of arabinan is normally about 15 kDa.
Arabinan-degrading enzymes are known to be produced by a variety of plants and micro-organisms. Three enzymes obtainable from A. niger have been cloned by molecular biological techniques (EPA 0506190). Also arabinosidase from bacteria such as Bacteroides has been cloned (Whitehead and Hespell (1990). J. Bacteriol. 172, 2408).
Galactomannans are storage polysaccharides found in the seeds of Leguminosae. Galactomannans have a linear (14)-xcex2-mannan backbone and are substituted with single (16)xcex1-galactose residues. For example in guar gum the ratio mannose/galactose is about 2 to 1. Galactomannans are applied as thickeners in food products like dressings and soups.
Mannanase enzymes are described in PCT application WO 93/24622.
Glucomannan consists of a main chain of glucose and mannose. The main chain may be substituted with galactose and acetyl groups; mannanases can be produced by a number of microorganisms, including bacteria and fungi.
To summarise, it can be said that a large number of plant cell wall degrading enzymes exist, produced by different organisms. Depending on their source the enzymes differ in substrate specificity, pH and temperature optima, Vmax, Km etc. The complexity of the enzymes reflects the complex nature of plant cell walls which differ strongly between plant species and within species between plant tissues. A suitable enzyme mixture can be prepared depending on the source of plant material, the purpose of the application and the specific application conditions.
In recent years the availability and variety of these cell wall degrading enzymes has increased considerably, which opens up the possibility of using selected combinations of these enzymes as additives in detergents. These detergents are particularly suitable for the removal of stains from vegetable origin.
Whereas the more thoroughly studied cell wall degrading enzymes originate from fungi and display pH optima in the acid pH range, nowadays more and more CWDE""s are being described which are active in more alkaline conditions, e.g.: xylanases (Shendye A, Rao M, 1993, FEMS Microbiol Lett 108, 297-302; Nakamura S, Wakabayashi K, Nakai R, Aono R, Horikoshi K, 1993, Appl Environ Microbiol 59, 2311-2316), mannanases (Akino T, Nakamura N, Horikoshi K, 1988, Agric Biol Chem 52, 773-779), galactanases (Tsumura K, Hashimoto Y, Akiba T, Horikoshi K, 1991, Agric Biol Chem 55, 125-127). This property makes these enzymes compatible with the current detergent formulations.
In many cases it will be possible to obtain the enzymes useful in the invention by culturing a micro-organism producing it and isolating the enzyme from the culture or the culture broth.
The enzymes useful in the invention can also be obtained through recombinant DNA technology, whereby a host cell is provided with the genetic information encoding the desired enzyme, together with suitable elements for expression of that genetic information.
A host cell may be a homologous micro-organism, or a heterologous micro-organism, which both may include but are not limited to bacteria, bacilli, yeasts and fungi; they can however also include higher eukaryotic cells such as plant or animal cells. It may also be very useful to provide a host cell with genetic information encoding more than one enzyme or more than one enzyme activity, for example a hybrid enzyme.
Although some emphasis has been placed on micro-organisms as a convenient source for the enzymes useful in the invention, it will be understood that enzymes from any source may be used, as long as they possess the activity of being able to break down at least parts of plant cell walls.
Since this activity is the most relevant property it will be clear that derivatives, fragments or combinations thereof with the same or similar activity can also be used and are to be included within the definition of enzyme.
Derivatives are explicitly meant to include mutants in which one or more amino acids have been added, deleted or substituted to maintain or improve certain properties of the enzymes, as well as chemically modified enzymes.
Compositions according to the invention may comprise a single enzyme (in which case the enzyme will not be a cellulase), although it is preferred that they contain a mixture of different enzymes, which are preferably capable of degrading different parts of plant cell walls or other components of stains, which stains have at least in part, a similar structure (e.g. stains of a food composition in which plant cell wall components are present as thickeners or gelating agents or the like).
For the most efficient removal of stains enzymes are preferred which have endo-splitting activities. These enzymes cut polymeric fibre compounds into smaller pieces and therefore increase the solubilization of the fibre mass with its associated pigments.
The compositions may be specifically adapted for their intended use. Compositions for cleaning textiles, either by hand or automatically will generally comprise different ingredients than compositions for cleaning kitchenware or for instance floors and tiles. Especially preferred compositions are so-called xe2x80x9cpre-spottersxe2x80x9d.
Usual ingredients for such compositions include surfactants, builders, bleaching agents, enzymes such as amylases and proteases, etc. The preferred compositions according to the invention are those intended for cleaning textiles.
Hence preferred compositions of the first aspect are detergent compositions. These may include washing powders and liquids, dish washing compositions, household or domestic (eg. floor and tile) cleaners, pre-wash compositions and/or other textile, fabric and cloth cleaning compositions.
A second aspect of the invention relates to a method of cleaning an object or surface, the method comprising contacting the object or surface with a composition of the first aspect and allowing cleaning to occur. The surface may be present on, for example, a floor or tile, and the object can be a textile or fabric article or an item of kitchenware (such as cutlery or crockery). Preferred features and characteristics of the second aspect are as for the first mutatis mutandis.
The invention will be explained in more detail in the following examples which are provided for illustration and are not to be construed as being limiting on the invention.